Inteins at Eleven Distinct Insertion Sites in Archaeal Helicase Subunit MCM Exhibit Varied Architectures and Activity Levels Across Archaeal Groups

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have produced a manuscript regarding the inteins of archeal bacteria.

The Introduction must be re-written, as the authors do not support well their case. Please, add a clear statement about the novelty and the originality of these findings. Also, please explain the advantage of this study over previous similar works. What gaps exactly in the international literature would be filled after publishing this manuscript?

M&M

Please add a separate sub-section describing in minute detail all the control material used in the study and all the control procedures followed. At the moment, this is unclear and points to methodological flaws.

Results

Please provide in supplementary material, a detailed table with all phylogenies generated for this work.

Visualisation is excellent.

I recommend to add more tables in the manuscript to make flow of reading easier.

Discussion

The Discussion is shallow.

I would have expected at least 3-4 pages of discussion in such an important topic. I was disappointed that the authors do not seem to make much use of their findings.

References. I expect at least 60-70 references for such a complicated and extensive study.

Conclusion

The concluding section must be rephrased to be in line with the actual findings of the work.

Overall. The recommendation is extensive modification and revision to improve the manuscript and resubmission.

Author Response

We would like to thank the reviewer for their feedback, and have provided a point-by-point response to their comments in the attached document: Author_Response_to_Reviewers.docx (see Reviewer 1 section)

Author Response:

Reviewer 1:

We would like to thank the reviewer for their time, and for their helpful feedback on this manuscript.

Comment 1: The Introduction must be re-written, as the authors do not support well their case. Please, add a clear statement about the novelty and the originality of these findings. Also, please explain the advantage of this study over previous similar works. What gaps exactly in the international literature would be filled after publishing this manuscript?

Response 1:

Introduction has been rephrased to emphasize the novelty of the intein architectures discovered through this work (see lines 133-137):

Our structural analyses revealed three sites within the MCM inteins where insertions resembling DNA-binding domains are found. These insertions vary in presence and size, adding a second facet to the inteins’ architectural diversity beyond the status of their homing endonucleases (mini or full). These insertions further expand the repertoire of known intein architectures.

Introduction has been rephrased to emphasize the novelty and importance of discovering full, mini, and empty intein alleles co-existing in a single group of geographically local archaeal populations (see lines 142-146)

Such a mixture of alleles has not yet been reported, and strongly supports the co-existence model of intein persistence and captures the varied histories of inteins found at the different sites of a multi-intein gene. Thus, these findings mark an important advancement in our understanding of intein evolutionary dynamics and persistence across interacting populations.

M&M

Comment 2:

Please add a separate sub-section describing in minute detail all the control material used in the study and all the control procedures followed. At the moment, this is unclear and points to methodological flaws.

Response 2:

This work is discovery driven by nature, thus standard controls one might have for hypothesis driven work do not apply. When we identify a molecular parasite, i.e., an intein, encoded inside an MCM gene, and this molecular parasite has the key amino acid residues involved in catalyzing the self-splicing reaction (and often also a homing endonuclease domain), the identity of the insertion sequence is beyond reasonable doubt. The closest equivalent to controls for this work are the many intein-free copies of the host protein sequence to which intein-containing sequences are compared to deduce intein-host protein boundaries (provided in Supplemental Data 1), and the solved protein structures to which predicted protein structures are compared when attempting to ascertain potential functions of different domains (said solved protein structures are included and cited in the legend of Figure 6 and its corresponding discussion under the Discussion section titled “Potential origin and role of the within-intein insertions”).

Results

Comment 3:

Please provide in supplementary material, a detailed table with all phylogenies generated for this work.

Response 3:

Supplemental Data 3 has now been added which contains the sequence alignments and tree files for all phylogenies generated for this work, and Supplemental Table S2 describes these phylogenies.

Comment 4:

I recommend to add more tables in the manuscript to make flow of reading easier.

Response 4:

In addition to the summary Table (Table 1) and the Tables in supplementary materials, we already had included tables in Figure 2A and 5, plus a histogram in Figure 4. We feel that additional tables would be repetitive. 

Discussion

Comment 5:

The Discussion is shallow.

I would have expected at least 3-4 pages of discussion in such an important topic. I was disappointed that the authors do not seem to make much use of their findings.

Response 5:

The Discussion has been expanded, particularly the portion titled “Archaeal MCM is a powerful system for the continued exploration of multi-intein gene dynamics.”

References.

Comment 6:

I expect at least 60-70 references for such a complicated and extensive study.

Response 6:

The number of citations has been increased.

Conclusion

Comment 7:

The concluding section must be rephrased to be in line with the actual findings of the work.

Response 7:

The Conclusions section follows the same structure followed throughout the rest of the work (MCM inteins overview, followed by novel intein architectures, followed by evidence supporting co-existence intein persistence model), directly reporting exact findings from each section. We begin by stating our discovery of six novel intein insertion sites in the minichromosome maintenance (MCM) gene of archaea (this is accurate to our findings). Next, we state our observation of high levels of variance in intein presence in MCM across archaea (this is accurate to our findings). Next, we state our discovery of insertions within these inteins which bear semblance to known DNA-binding domains (this is accurate to our findings). Lastly, we state our discovery of a group of archaeal populations with full, mini, and empty intein alleles co-existing which lends novel and strong support to co-existence models of intein persistence (this is accurate to our findings).

Reviewer 2 Report

Comments and Suggestions for Authors

The reviewer has provided the following comments for further improvement of the quality, clarity and understanding of the manuscript.

Comments:

• At the end of the abstract: Add a brief statement on the major implication/contribution of this work.
• Line 74: “intein architectures and evolutionary behaviors, particularly in archaea.” Why you are interested in studying on the prevalence of intein in only archaeal communities? Were they not explored in past studies?
• Line 80: “archaeal gene mcm, which encodes the MCM subunit of replicative DNA helicase…”. Explain the abbreviation “MCM” which may not be known to the non-expert readers.
• To better understand the background information, authors are suggested to add important findings from the 2 – 3 recent publications related to this study.
• Introduction: Overall, it is highly qualitative in nature at present. Improve it by adding quantitative data.
• Line 120 – 121: “, PSI-BLAST [19] searches were performed against…”. Give full name of “PSI-BLAST”.
• Before conclusions: Include a section describing on the “Important implications and future perspectives of this study”
• To enhance readability of the manuscript, authors should consider explain the abbreviations which is not well-known to wider readers at their first appearance in the text as well as provide brief statement about the function and importance of the reported genes (e.g., polB).
• Figures: Make sure the reported figure are in high quality, i.e., high resolution (>300 dpi), the font size of the text is well enough to visible, the color combinations should be wisely selected with discouraging to use light color, etc.

Author Response

We would like to thank the reviewer for their feedback, and have provided a point-by-point response to their comments in the attached document: Author_Response_to_Reviewers.docx (see Reviewer 2 section)

Author Response:

Reviewer 2:

We would like to thank the reviewer for their time, and for their helpful feedback on this manuscript.

Comment 1:

At the end of the abstract: Add a brief statement on the major implication/contribution of this work.

Response 1:

We have tightened up the Conclusions portion at the end of our Abstract to more concisely state the importance of our findings (see lines 33-40):

Conclusions: We identified six new active intein insertion sites in archaeal MCM, more than doubling the five previously known sites. All eleven intein insertion sites were either close to the ATP binding site, or the lined the channel through which the single stranded DNA is pulled during the catalytic cycle of the helicase. Many of the analyzed inteins contained insertions bearing similarity to DNA-binding helix-turn-helix domains suggesting potential involvement in the intein homing process. Additionally, the high levels of MCM intein diversity observed in archaea from the Atacama Desert provide novel and strong support for a co-existence model of intein persistence.

Comment 2:

Line 74: “intein architectures and evolutionary behaviors, particularly in archaea.” Why you are interested in studying on the prevalence of intein in only archaeal communities? Were they not explored in past studies?

Response 2:

Introduction has been rephrased to emphasize the importance of studying archaea. They are the most understudied domain of life, and engage in high levels of horizontal gene transfer. Studying their inteins in particular is the focus of this particular work (though our research group also studies inteins in non-archaeal systems, covered in other works), and helps shed light on the pathways for horizontal transfer available between different archaeal populations (see lines 88-94):

Archaea, which experience high levels of horizontal transfer [10], [11], [12], contain inteins in a wide range of genes [13]; however, the majority of intensive archaeal intein characterization efforts have been focused on a few select groups such as haloarchaea. In addition to being an understudied group ripe for the exploration of intein architectures and evolutionary dynamics, archaea offer a unique area of intein exploration in which both architecture and evolution can be studied: genes invaded by multiple inteins simultaneously [14], [15].

Comment 3:

Line 80: “archaeal gene mcm, which encodes the MCM subunit of replicative DNA helicase…”. Explain the abbreviation “MCM” which may not be known to the non-expert readers.

Response 3:

Thank you for catching this, in the text we now provide the full name before the abbreviation (minichromosome maintenance (MCM)) at its first mention (line 12 in Abstract, also line 95 in Introduction).

Comment 4:

To better understand the background information, authors are suggested to add important findings from the 2 – 3 recent publications related to this study.

Response 4:

Recent associated works and findings are covered in the following section of the Introduction, with citation 15 (Turgeman-Grott et al. 2023) being the most directly and recently related to this work (see lines 94-121):

The archaeal gene mcm, which encodes the minichromosome maintenance (MCM) subunit of replicative DNA helicase [16], [17], contains five intein insertion sites named MCM-a through e respectively. Inteins at sites MCM-a through d have been the subject of intein insertion site recognition and self-splicing investigations, particularly in haloarchaea [15], [18]. Insertion site MCM-e is not invaded in any haloarchaea analyzed to date, but the site is active in some groups of non-haloarchaea. The MCM-e intein published to the intein database InBase 2.0 [19] in 2012 was from Thermococcus litoralis, with the in-sertion site name CDC21-e [20]. An analysis of the MCM inteins at sites MCM-a through d in haloarchaeal MCM homologs from the order Haloferacales revealed a wide array of intein invasion statuses (empty, single, double, triple, and quadruple), mini and full inteins at the same insertion sites in different homologs, and sporadic distribution of the four inteins across the host protein phylogeny [15]. The diversity of MCM inteins in this order alone begs the question of whether such patterns will hold when a similar analysis is performed on other archaeal lineages, and whether such diversity can also be found in a single group of geographically overlapping populations of archaea as opposed to a mass sampling of sequences from a wide array of timepoints and geographic locations.

Introduction

Comment 5:

Overall, it is highly qualitative in nature at present. Improve it by adding quantitative data.

Response 5:

Quantitative values now included in the Introduction include:

• Number of MCM homologs investigated (4,243)
• The new total of known MCM intein insertion sites (5 were known previously, and we discovered 6 new sites through our work, raising the total to 11)
• The number of unique MCM intein architectures we observed (17)
• Observed range of within-intein insertion sizes (20 amino acids (aa) to 200 aa)

Comment 6:

Line 120 – 121: “, PSI-BLAST [19] searches were performed against…”. Give full name of “PSI-BLAST”.

Response 6:

Thank you for catching this, the full name is now provided in this line (now line 151) and in the Abbreviations section (line 540).

Comment 7:

Before conclusions: Include a section describing on the “Important implications and future perspectives of this study”

Response 7:

This has been provided for each major section covered in the Discussion:

Regarding MCM as a system for intein studies (lines 434-438):

Thus, the six additional MCM intein insertion sites presented in this work offer new avenues for exploring molecular and evolutionary dynamics between inteins co-inhabiting a gene. In addition to these newly discovered intein insertion sites, the range of MCM intein architectures discovered opens avenues for further investigation of the biochemical versatility of inteins with additional, potentially DNA-binding, domains.

Regarding the within-intein insertions (lines 462-465):

Thus, it is possible that the insertions within the MCM inteins are involved in the homing process, potentially in stabilizing the binding of the intein to its target DNA. Future work can build on our findings to assess the individual roles of each of the three types of within-intein insertions of MCM inteins.

Regarding support for co-existence model of intein persistence (lines 491-496):

Thus, the MCM inteins in these populations operate in a manner more in line with a co-existence model for intein persistence [7], [8], [9], rather than the synchronized Goddard-Burt life cycle [5]. Future investigations of intein dynamics within geographically overlapping populations will continue to shed light on the frequencies of co-existence versus synchronized progression modes for intein persistence in natural populations.

Comment 8:

To enhance readability of the manuscript, authors should consider explain the abbreviations which is not well-known to wider readers at their first appearance in the text as well as provide brief statement about the function and importance of the reported genes (e.g., polB).

Response 8:

Abbreviations lacking definitions have now been defined upon first mention in the text and in the Abbreviations section, and a brief description of polB and bacterial ribonucleotide reductase have been provided (lines 398-402):

The archaeal gene polB, encoding B-type DNA polymerase which is involved in DNA replication [40], [41], has up to three inteins simultaneously invading investigated copies of the gene [14]. Additionally, a gene encoding a bacterial ribonucleotide reductase (RIR), an important enzyme for DNA synthesis [42], [43], has up to four inteins in investigated copies [44].

Comment 9:

Figures: Make sure the reported figure are in high quality, i.e., high resolution (>300 dpi), the font size of the text is well enough to visible, the color combinations should be wisely selected with discouraging to use light color, etc.

Response 9:

Each figure has been reviewed and re-exported with higher quality (> 300 dpi)

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

Authors have addressed and included most of the comments in the revised version.